DESCRIPTION (Applicant's Description): The long-term goal of this project is to determine the function of general transcription factor IIA (TFIIA). TFIIA is required to mediate activator-stimulated transcription in human cell-free reactions. Two different human viruses, Epstein Barr virus (EBV) and human T-cell lymphotropic virus type 1 (HTLV-1), encode proteins which activate transcription by associating with TFIIA. The specific goal of this application is to investigate the role of TFIIA in mediating activator-stimulated transcription in vivo. TFIIA stimulates TATA binding protein (TBP)-promoter interactions in vitro, which has been proposed to be a limiting step in the activation of some mRNA expressing genes in vivo. The TFIIA genes are highly conserved between yeast and human, and the ability of TFIIA to stimulate TBP promoter binding is similarly conserved. Both biochemical and genetic approaches will be used to study how TFIIA functions in vivo using the budding yeast Saccharomyces cerevisiae as a model system. Mutations in TFIIA that compromise its ability to bind TBP have been generated, and are conditionally lethal in S. cerevisiae. These mutants will be used to determine whether the TFIIA-TBP interaction is rate-limiting for all class II gene transcription or a subset of genes. TFIIA may mediate the transcriptional activity of a subset of activators or genes that contain a particular promoter structure. Preliminary work has shown that these TFIIA mutants accumulate in G2/M phase of the cell division cycle. The applicants will determine if G2/M specific gene transcription is particularly sensitive to the TFIIA-TBP interaction. They have isolated a high copy suppressor of TFIIA growth arrest. A conserved Drosophila homologue of this gene, pelota, has been implicated in G2/M progression. The applicants propose to characterize the genetic and biochemical basis of pelota suppression of TFIIA transcriptional defects. This proposal will provide important new information concerning transcriptional activation in eukaryotic organisms and will specifically address the role of TFIIA in regulating viral and cell cycle dependent transcription.